Passage procedures for epithelial cells showing strong cell-to-cell contact (like MDCK cells and NRK-52E cells)
Aim
Passage procedures for cultured cells
Materials
Medium (e.g., DMEM, 10% FBS)
PET (PBS/0.01% EDTA/0.25% Trypsin) solution
(For cells with strong cell-to-cell contact such as MDCK cells) PBS/1 mM EDTA
Methods
(Important) Before starting the following steps, you must check cell condition by microscope, such as confluency, morphology, etc.
Warm medium, PET solution and PBS/1 mM EDTA solution to 37 degrees.
Move medium, PET solution, PBS/1 mM EDTA solution and culture cell dish into safety cabinet.
Aspirate the medium in the culture dish.
Add 7-10 mL PBS/1 mM EDTA to the dish, and incubate the dish for 5 min in a humidified CO2 incubator.
This process allows attenuating cell-to-cell contact of the cells.
Aspirate the PBS/1 mM EDTA solution in the culture dish.
Add 1 mL PET solution to the dish, spread the PET solution throughout the surface of the dish.
Aspirate the PET solution, and incubate the dish for 3-5 min in the humidified CO2 incubator.
The tiny amount of PET solution covering the surface on culture dish is enough to detach cells from the dish.
This process also reduces trypsin carry-over.
(Important) Check whether cells are really detached from the dish under a microscope.
Suspend the cells with the serum-containing medium by pipetting with 10 mL disposable pipet.
Residual trypsin must be inhibited by serum.
Split the suspended cells into new culture dishes with the new medium.
MDCK cells: 1:6 and 1:12 ratio for 2 and 3 days culture, respectively.
Remarks
Date : 2018-03-15
Modified Date :
Author : gmaryu.icon
Passage of cultured cells (like HeLa and 293T cells) (copy)